酿酒酵母β-葡聚糖通过膜受体Dectin-1和TLR-2诱导绵羊瘤胃上皮细胞表达SBD-1的机制研究
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摘要
为了探索酿酒酵母β-葡聚糖诱导绵羊瘤胃上皮细胞(ovine ruminal epithelial cells,ORECs)β-防御素-1(sheepβ-defensin-1,SBD-1)表达过程中膜受体Dectin-1和TLR-2可能存在的作用。本研究首先利用免疫组化、RT-PCR、免疫荧光和Western blot等方法检测Dectin-1是否在ORECs内表达,并且采用qPCR和Western blot方法对β-葡聚糖刺激ORECs后细胞膜受体Dectin-1和TLR-2的表达变化进行检测。而后用不同浓度的Dectin-1阻断剂昆布多糖或TLR-2特异性封闭抗体分别预处理ORECs后,采用qPCR和ELISA方法检测SBD-1的表达变化,以确定β-葡聚糖诱导SBD-1表达过程中Dectin-1和TLR-2的参与情况。结果显示:1)绵羊瘤胃组织及ORECs内存在Dectin-1表达,且β-葡聚糖刺激ORECs后Dectin-1和TLR-2的表达水平显著增加(P<0.05);2)不同浓度的昆布多糖和TLR-2封闭抗体均可以极显著降低β-葡聚糖诱导SBD-1的表达(P<0.01),且随着阻断剂和封闭抗体浓度的增加,其抑制SBD-1表达的作用越明显。结果表明,Dectin-1在绵羊瘤胃组织及ORECs内均表达,并且酿酒酵母β-葡聚糖在ORECs中诱导SBD-1的表达是由Dectin-1和TLR-2介导产生的。
The aim of this study was to explore the possible role of TLR-2 and Dectin-1 in the expression of β-defensin-1(SBD-1) in ovine ruminal epithelial cells(ORECs) induced by Saccharomyces cerevisiae β-glucan. The immunohistochemistry, RT-PCR, immunofluorescence and Western blot were used to detect the expression of Dectin-1 in ORECs, and qPCR and Western blot were used to detected the expression changes of Dectin-1 and TLR-2 after β-glucan stimulated ORECs. Then, ORECs were pretreated using Dectin-1 blocker laminarin or TLR-2 blocking antibody with different concentrations, and the expression of SBD-1 were detected by qPCR and ELISA to determine the involvement of Dectin-1 and TLR-2 in the induction expression of SBD-1 induced by β-glucan. The results showed that: 1) Dectin-1 was expressed in ovine ruminal tissues and ORECs, and the expression levels of Dectin-1 and TLR-2 were significantly increased after β-glucan stimulating ORECs(P<0.05); 2)The laminarin or TLR-2 blocking antibody with different concentrations significantly decreased the expression of SBD-1 induced by β-glucan(P<0.01), and this effect became stronger as the laminarin and blocking antibodies concentration increased. The results indicate that Dectin-1 is expressed in both ovine ruminal tissues and ORECs, and the Saccharomyces cerevisiae β-glucan inducing SBD-1 expression in ORECs is mediated by Dectin-1 and TLR-2.
The aim of this study was to explore the possible role of TLR-2 and Dectin-1 in the expression of β-defensin-1(SBD-1) in ovine ruminal epithelial cells(ORECs) induced by Saccharomyces cerevisiae β-glucan. The immunohistochemistry, RT-PCR, immunofluorescence and Western blot were used to detect the expression of Dectin-1 in ORECs, and qPCR and Western blot were used to detected the expression changes of Dectin-1 and TLR-2 after β-glucan stimulated ORECs. Then, ORECs were pretreated using Dectin-1 blocker laminarin or TLR-2 blocking antibody with different concentrations, and the expression of SBD-1 were detected by qPCR and ELISA to determine the involvement of Dectin-1 and TLR-2 in the induction expression of SBD-1 induced by β-glucan. The results showed that: 1) Dectin-1 was expressed in ovine ruminal tissues and ORECs, and the expression levels of Dectin-1 and TLR-2 were significantly increased after β-glucan stimulating ORECs(P<0.05); 2)The laminarin or TLR-2 blocking antibody with different concentrations significantly decreased the expression of SBD-1 induced by β-glucan(P<0.01), and this effect became stronger as the laminarin and blocking antibodies concentration increased. The results indicate that Dectin-1 is expressed in both ovine ruminal tissues and ORECs, and the Saccharomyces cerevisiae β-glucan inducing SBD-1 expression in ORECs is mediated by Dectin-1 and TLR-2.
引文
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[2] O′SHEA E F,COTTER P D,STANTON C,et al.Production of bioactive substances by intestinal bacteria as a basis for explaining probiotic mechanisms:bacteriocins and conjugated linoleic acid[J].Int J Food Microbiol,2012,152(3):189-205.
[3] KLASING K C.Nutritional modulation of resistance to infectious diseases[J].Poult Sci,1998,77(8):1119-1125.
[4] KLASING K C.Nutrition and the immune system[J].Br Poult Sci,2007,48(5):525-537.
[5] KIDD M.Nutritional modulation of immune function in broilers[J].Poult Sci,2004,83(4):650-657.
[6] BERGE A C,WIERUP M.Nutritional strategies to combat Salmonella in mono-gastric food animal production[J].Animal,2012,6(4):557-564.
[7] CASADEI E,WANG T H,ZOU J,et al.Characterization of three novel β-defensin antimicrobial peptides in rainbow trout (Oncorhynchus mykiss)[J].Mol Immunol,2009,46(16):3358-3366.
[8] ELLIS A E.Innate host defense mechanisms of fish against viruses and bacteria[J].Dev Comp Immunol,2001,25(8-9):827-839.
[9] GANZ T,LEHRER R I.Antimicrobial peptides of vertebrates[J].Curr Opin Immunol,1998,10(1):41-44.
[10] OUELLETTE A J.Paneth cell α-defensin synthesis and function[M]//SHAFER W M.Antimicrobial Peptides and Human Disease.Berlin,Heidelberg:Springer,2006,306:1-25.
[11] SCHLEE M,HARDER J,K?TEN B,et al.Probiotic lactobacilli and VSL#3 induce enterocyte β-defensin 2[J].Clin Exp Immunol,2008,151(3):528-535.
[12] KUBOTA A,KOBAYASHI M,SARASHINA S,et al.Reishi mushroom Ganoderma lucidum Modulates IgA production and alpha-defensin expression in the rat small intestine[J].J Ethnopharmacol,2018,214:240-243.
[13] ABDELSALAM M,ISOBE N,YOSHIMURA Y.Effects of lipopolysaccharide and interleukins on the expression of avian β-defensins in hen ovarian follicular tissue[J].Poult Sci,2012,91(11):2877-2884.
[14] VOLMAN J J,RAMAKERS J D,PLAT J.Dietary modulation of immune function by β-glucans[J].Physiol Behav,2008,94(2):276-284.
[15] QI C J,CAI Y H,GUNN L,et al.Differential pathways regulating innate and adaptive antitumor immune responses by particulate and soluble yeast-derived β-glucans[J].Blood,2011,117(25):6825-6836.
[16] SCHMITT P,WACYK J,MORALES-LANGE B,et al.Immunomodulatory effect of cathelicidins in response to a β-glucan in intestinal epithelial cells from rainbow trout[J].Dev Comp Immunol,2015,51(1):160-169.
[17] SHAO Y J,WANG Z,TIAN X Y,et al.Yeast β-D-glucans induced antimicrobial peptide expressions against Salmonella infection in broiler chickens[J].Int J Biol Macromol,2016,85:573-584.
[18] CAMPOVERDE C,MILNE D J,ESTéVEZ A,et al.Ontogeny and modulation after PAMPs stimulation of β-defensin,hepcidin,and piscidin antimicrobial peptides in meagre (Argyrosomus regius)[J].Fish Shellfish Immunol,2017,69:200-210.
[19] BROWN G D,GORDON S.Immune recognition.A new receptor for β-glucans[J].Nature,2001,413(6851):36-37.
[20] WILLMENT J A,GORDON S,BROWN G D.Characterization of the human β-glucan receptor and its alternatively spliced isoforms[J].J Biol Chem,2001,276(47):43818-43823.
[21] HARDISON S E,BROWN G D.C-type lectin receptors orchestrate antifungal immunity[J].Nat Immunol,2012,13(9):817-822.
[22] SUKHITHASRI V,NISHA N,BISWAS L,et al.Innate immune recognition of microbial cell wall components and microbial strategies to evade such recognitions[J].Microbiol Res,2013,168(7):396-406.
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[34] FUKATA M,ARDITI M.The role of pattern recognition receptors in intestinal inflammation[J].Mucosal Immunol,2013,6(3):451-463.
[35] NALUBAMBA K S,GOSSNER A G,DALZIEL R G,et al.Differential expression of pattern recognition receptors in sheep tissues and leukocyte subsets[J].Vet Immunol Immunopathol,2007,118(3-4):252-262.
[36] DE SMET R,DEMOOR T,VERSCHUERE S,et al.β-Glucan microparticles are good candidates for mucosal antigen delivery in oral vaccination[J].J Control Release,2013,172(3):671-678.
[37] KOLAR S S,BAIDOURI H,MCDERMOTT A M.Role of pattern recognition receptors in the modulation of antimicrobial peptide expression in the corneal epithelial innate response to F.solani[J].Invest Ophthalmol Vis Sci,2017,58(5):2463-2472.
[38] ILIEV I D.Dectin-1 exerts dual control in the gut[J].Cell Host Microbe,2015,18(2):139-141.
[39] LEE J Y,KIM J Y,LEE Y G,et al.Molecular mechanism of macrophage activation by exopolysaccharides from liquid culture of Lentinus edodes[J].J Microbiol Biotechnol,2008,18(2):355-364.